CN108486065A - Secrete hybridoma cell strain and its monoclonal antibody application of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody - Google Patents
Secrete hybridoma cell strain and its monoclonal antibody application of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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Abstract
The invention discloses a kind of applications of the hybridoma cell strain and its monoclonal antibody of the anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody of secretion.It is that BALB/c mouse is immunized in antigen with Rice Gall Dwarf In Guangdong Province (RGDV) CP recombinant proteins of prokaryotic expression, through cell fusion, screening, clone, the hybridoma cell strain 11B6 for passing on and secreting anti-RGDV monoclonal antibodies can be stablized by obtaining 1 plant, and preserving number is CGMCC No.14897.The odd contradictive hydroperitoneum indirect ELISA titer of hybridoma cell strain secretion reaches 10‑8, Antibody types and subclass are IgG1, kappa.The monoclonal antibody has specific reaction with RGDV coat protein.It is ACP ELISA and the dot ELISA methods that core establishes detection RGDV using 11B6 monoclonal antibodies, the sensitivity of the sick leaf of both methods detection respectively reaches 1:327 680 and 1:20 480 times of dilutions (w/v, g/mL).The acquisition of the hybridoma cell strain and its monoclonal antibody and the detection, diagnosis and science bridle for being established as the virosis of Serology test provide substance and technical support.
Description
Technical field
The present invention relates to biotechnology more particularly to a kind of hybridization of the anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody of secretion
The application of tumor cell strain and its monoclonal antibody.
Background technology
Rice gall dwarf virus disease is a kind of rice caused by Rice Gall Dwarf In Guangdong Province (Rice gall dwarf virus, RGDV)
Virus disease.From 1979 after Thailand finds, rice gall dwarf virus disease is found on Malaysia, Japan, South Korea and other places in succession.1981
Year finds RGDV for the first time in China Guangdong, and rice gall dwarf virus disease Major Epidemic is in the Guangdong in China, Guangxi, Fujian, Hainan etc. at present
South Rice Region of China, serious field of falling ill even can not normally ear, and No kernels or seeds are gathered, as in a year of scarcity.RGDV host plants are also wrapped other than rice
The gramineous crops such as wheat, barley, oat, amur foxtail, wild rice and corn are included, but it does not infect barnyard grass.Infect RGDV rice
Classical symptom include：The plant of morbidity stunts, and tiller is reduced, and young leaves is short and narrow, and leaf color deepens, and blade back portion and leaf sheath have 0.1-
The circular white strumae of 0.2mm sizes has 0-30 per leaf.RGDV is by vector recilia dorsalis and rice green leafhopper to hold
Long proliferous type mode is propagated, and leafhopper once obtains poison, i.e., lifelong band poison simultaneously can transovarian transmission.RGDV belongs to Reoviridae
(Reoviridae), Phytoreovirus (Phytoreovirus).The virion of RGDV belongs to icosahedron, triple
Symmetrically, spherical, diameter is about 65-70nm.The genome sequence of RGDV is all measured and is finished, and is formed containing 12 dsRNA,
Every RNA segment only encode an albumen, including six structural proteins (P1, P2, P3, P5, P6 and P8) and six it is non-structural
Albumen (Pns4, Pns7, Pns9, Pns10, Pns11 and Pns12).
Recilia dorsalis and rice green leafhopper are China is widely distributed, harmfulness is big, and it draws as passing virus mediator and propagate RGDV
The rice gall dwarf virus disease risen is serious in China's harm in recent years, causes Severe Reduction of Rice.And the detection technique of virus is the virus
The key of sick prevention and control.Field rice tumor dwarf virus is judged a situation arises to be still most important side according to Symptom Observation at present
Method, but virus infection rice early period does not have any symptom, and symptom caused by a variety of virosis is very much like, in addition, often ill
Malicious Combined Infection situation, therefore lack accuracy and science by the result that Symptom Observation obtains.Electronic Speculum observes virion
Expensive instrument is needed, and the size of somewhat different virion and form have similitude, it is caused only to be examined as a kind of auxiliary
Survey means.The molecular detection technology of based on PCR method is although sensitive, but is unsuitable for the detection of the extensive sample in field.Serum
Method is simple to operation, quick, sensitive, cost is relatively low, and suitable for the extensive detection of field sample, is ideal at present
The detection technique of plant virus, but serological method is necessarily dependent upon special, sensitive high-quality antibody.For this purpose, patent of the present invention
Preparation and its detection application mainly for Rice Gall Dwarf In Guangdong Province monoclonal antibody are carried out the work, and are prepared for using hybridoma technology
1 plant can secrete anti-RGDV monoclonal antibodies hybridoma 11B6, its monoclonal antibody secreted is used in combination to establish the serology side for detecting the virus
Method, to provide substance and technical support for the detection, diagnosis and science bridle of China's Rice Gall Dwarf In Guangdong Province.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody of secretion
The application of hybridoma cell strain and its monoclonal antibody.
Anti- Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma 11B6 is secreted, the specificity that it can secrete anti-Rice Gall Dwarf In Guangdong Province is single
Anti-, hybridoma cell strain 11B6 was preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on December 15th, 2017
Object center, preserving number are CGMCC No.14897.
The monoclonal antibody ascites indirect ELISA titer of anti-Rice Gall Dwarf In Guangdong Province is up to 10-8, Antibody types and subclass are
The coat protein of IgG1, kappa, the monoclonal antibody and Rice Gall Dwarf In Guangdong Province 47kDa has specific immune response, utilizes ACP-
ELISA and dot-ELISA method analyses find that the sensitivity of monoclonal antibody detection infection RGDV rice disease leaves respectively reaches 1:327
680 and 1:20 480 times of dilutions (w/v, g/mL).
The monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province only has a specific immune response with Rice Gall Dwarf In Guangdong Province, and with rice item
Line virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, rice stripe mosaic disease
It poison and healthy rice plants and non-take malicious recilia dorsalis and non-take malicious rice green leafhopper tissue any immune response does not occur.
Application of the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province in Rice Gall Dwarf In Guangdong Province detection is built by core of monoclonal antibody
Vertical various immunological detection methods and immunological detecting kit.
The present invention has the advantage that compared with prior art：1) the hybridoma cell strain 11B6 provided secretes water resistant rice
Tumor dwarf virus monoclonal antibody specific, the immunological methods such as the dot-ELISA and ACP-ELISA that are established using the monoclonal antibody as core
And established with these methods kit energy high special, it is accurate, delicately detect Rice Gall Dwarf In Guangdong Province；2) institute of the present invention is utilized
The monoclonal antibody of preparation detects Rice Gall Dwarf In Guangdong Province, does not need the equipment such as expensive electron microscope, PCR instrument；3) institute of the present invention is utilized
The monoclonal antibody of preparation is effectively used for the detect and diagnose of the internal water RGDV such as field rice and biography virus mediator recilia dorsalis, also may be used
For the epidemiological survey of the virosis, viral genome functional analysis, resistance breeding, science bridle etc..
Description of the drawings
Fig. 1 is the sensitivity analysis of dot-ELISA methods detection rice RGDV；CK+ is that the rice disease leaf of infection RGDV is thick
Extract, CK- are healthy rice leaf crude extract.
Fig. 2 is the specificity analysis of dot-ELISA methods detection RGDV：2 points are 1 infection RGDV rice to 1 row up and down
2 biology of plant sample repeat；2 points are the 2 biology repetition for recombinantly expressing RGDVCP albumen to 2 row up and down；On 3 row
Lower 2 points are that 2 biology of 1 infection RSV rice plants sample repeat；2 points are 1 infection RBSDV rice to 4 row up and down
2 biology of plant sample repeat；2 points are that 2 biology of 1 infection RRSV rice plants sample repeat to 5 row up and down；
2 points are that 2 biology of 1 infection SRBSDV rice plants sample repeat to 6 row up and down；2 points are 1 RSMV to 7 row up and down
2 biology of rice plants sample repeat；2 points are that 2 biology of 1 healthy rice plants sample repeat to 8 row up and down.
Fig. 3 is the representative result of RGDV in dot-ELISA methods detection rice field sample.
Biological deposits
It secretes anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma 11B6 and is preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, preservation day is 2017
December 15, preserving number are CGMCC No.14897.
Specific implementation mode
It secretes anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody hybridoma cell strain 11B6 and is preserved in Chinese science on December 15th, 2017
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of institute, preserving number CGMCC
No.14897, it can secrete the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province.
The monoclonal antibody ascites indirect ELISA titer of anti-Rice Gall Dwarf In Guangdong Province is up to 10-8, Antibody types and subclass are
IgG1, kappa can have specific immune response with the 47kDa coat protein of Rice Gall Dwarf In Guangdong Province, using ACP-ELISA and
Dot-ELISA method analyses find that the sensitivity of monoclonal antibody detection infection RGDV rice plants disease leaves respectively reaches 1:327 680
With 1:20 480 times of dilutions (w/v, g/mL).
The monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province only has a specific immune response with Rice Gall Dwarf In Guangdong Province, and with rice item
Line virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, rice stripe mosaic disease
It poison and healthy rice plants and non-take malicious recilia dorsalis and non-take malicious rice green leafhopper tissue any immune response does not occur.
Application of the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province on the viral diagnosis be established using monoclonal antibody as core it is various immune
Learn detection method and immunological reagent box.
Hybridoma cell strain 11B6 provided by the invention can largely secrete the monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province, and its secretion
Monoclonal antibody potency height, high specificity, high sensitivity, stability are good.The detection RGDV's established using the monoclonal antibody as core is high-throughput
Serological method can be successfully applied to the detection of field RGDV, to for the detect and diagnose of China's Rice Gall Dwarf In Guangdong Province, early warning,
Breeding for disease resistance and science bridle provide substance and technical support.
With reference to embodiment and attached drawing, the invention will be further described.
One, the acquisition of hybridoma and its preparation of monoclonal antibody
1. immunogene and the preparation for detecting antigen
According to the gene order (accession number for the RGDV coded housing albumen reported：D13410.1) design a pair is specifically drawn
Object：RGDV-CP-F:5’-CTGGTTCCGCGTGGATCCATGTCGCGCCAAGCTT-3 ' and RGDV-CP-R:5’-
GATGCGGCCGCTCGAGTCGACTTAGTTTACTGTGTAATACCTACCG-3 ', underscore part are BamH I, I digestions of Sal
Site, and synthesized by Hangzhou Qing Ke Bioisystech Co., Ltd.The total serum IgE of infection RGDV rice disease leaves is extracted with TRIzol reagents
(concrete operations are carried out with reference to reagent specification) has cloned the disease using RT-PCR method from the rice total RNA of infection RGDV
The CP genes of poison.
RT-PCR reacts：It is carried out according to the RNA reverse transcription reagent box specifications of mill company of Japan as follows：In a centrifuge tube
The middle 1 μ L of total serum IgE that 5 μ L of deionized water, 4 × DN Master Mix, 2 μ L, 65 DEG C of metal baths are added and are denaturalized 5min, 37 DEG C of digestion
Genomic DNA 5min；Add 2 μ L 5 × RT Master MixII, 37 DEG C of reverse transcription 1h after mixing；After 98 DEG C of inactivation 5min
It is immediately placed on ice, sample can save in -20 DEG C of refrigerators.PCR reaction systems (50 μ L) are as follows：dNTP 5μL、MgSO4 4μL、
5 μ L of KOD plus neo buffer, each 1 μ L of upstream and downstream primer, 1 μ L of cDNA templates, 33 μ L of deionized water.PCR response procedures
It is as follows：94 DEG C of pre-degeneration 2min, 98 DEG C of denaturation 10sec, 55 DEG C of annealing 30sec, 68 DEG C of extension 40sec, 68 DEG C after 32 cycles
Extend 10min, 16 DEG C of 5min.After reaction, it carries out the detection of PCR product with agarose gel electrophoresis and is tapped and recovered.
Recombinant expression carrier is built using the homologous recombination kit of Nuo Weizan companies：Reaction system (10 μ L) is：5×CE
2 μ L of II Buffer, 1 μ L of RGDV CP genetic fragments, 1.5 μ L of carrier, 1 μ L of Exnase II, ddH2O 4.5μL.According to above-mentioned
After evenly mixing, 37 DEG C of 30min are put on ice system later.Protokaryon table is inserted it into using the method for above-mentioned homologous recombination
Up to being built into recombinant prokaryotic expression vector pGEX-4T-3-CP in vector pGEX -4T-3.PGEX-4T-3- containing recombinant expression carrier
BL21-CodonPlus (DE3) Escherichia coli of CP obtain through IPTG induction expression proteins, GST-beads affinity column protein purifications
RGDV CP recombination fusion proteins.BALB/c mouse is immunized by antigen of the recombinant protein of purifying.
2. immune animal
With the 8 week old BALB/c female mices of RGDV CP protein immunizations of purifying.That is 80 μ L of RGDV purifying proteins and 80 μ L good fortune
Family name's Freund's complete adjuvant mixes, it is fully emulsified after through 160 μ L/ of intraperitoneal injection only, be spaced 3 weeks, take the antigen for exempting from difference equivalent with one and
After freund 's incomplete adjuvant is fully emulsified, intraperitoneal injection two exempt from, with 120 μ L/ antigens and 80 μ L/ after 3 weeks
Physiological saline mixing containing mycillin through intraperitoneal injection three exempt from, and three are repeated once after 2 weeks exempt from step and carry out reinforcing exempting from
Epidemic disease, extracting spleen cell is merged after 3 days.
3. cell fusion
Take above-mentioned immune mouse spleen cell with murine myeloma cell (SP2/0) by 6:RPMI- of 1 ratio in serum-free
Mixing in 1640 culture mediums, 1 500rpm centrifuge 5min and remove culture medium, use 50%PEG (molecular weight 1 500) as fusion agent,
2min is merged in 37 DEG C of water-baths, 1 500rpm centrifuges 5min, precipitation after terminating fusion with the RPMI-1640 culture mediums of serum-free
It is suspended, is dispensed into 96 porocyte plates with HAT culture mediums, be placed in 37 DEG C and contain 5%CO2Cell incubator in cultivate.
4. hybridoma, the screening in positive hole and its clone
After cultivating 5d in cell incubator, is changed the liquid once with HAT culture mediums, change liquid, Deng Daorong with HT culture mediums within the 10th day
When closing cell covering bottom hole 10-30%, using RGDV CP as antigen coat elisa plate, it is single to screen secretion with indirect ELISA method
80 positive holes are obtained in anti-positive hole altogether.Selection 8 is in the cell hole of strong positive reaction, carries out 3 limiting dilution assay clones,
Obtain 1 plant of hybridoma cell strain 11B6 that can secrete anti-RGDV specific monoclonal antibodies.It freezes through 6 months or more subculture in vitro separately and repeatedly
After recovery, cell strain can well be grown, and stably excreting antibody.After expanding and cultivating, for ascites preparation and Liquid nitrogen storage.
5. monoclonal antibody ascites prepares and purifying
(1) 8 week old or so BALB/c mouse is taken, 0.3mL norphytanes, 7-10d pneumoretroperitoneums injection 6 × 10 is injected intraperitoneally5It is a
Hybridoma, the visible mouse web portions of 7-10d obviously expand after injection, and syringe needle takes ascites, 3 000rpm to centrifuge 3min, collects
Supernatant is odd contradictive hydroperitoneum.
(2) saturated ammonium sulfate method purified monoclonal antibody ascites (by taking 1mL as an example)
1) 5 000rpm of 1mL ascites, 4 DEG C of centrifugation 5min are taken, supernatant is taken, 2mL normal saline dilutions is added；
2) pH value is added dropwise in the saturated ammonium sulfate solution 3mL of 7.0-7.4, stirring while adding, room temperature after being all added
Continue to stir 10min；4 DEG C of static 2-3h；
3) 12 000rpm, 4 DEG C of centrifugation 10min abandon supernatant, precipitation 2mL physiological saline solutions；
4) antibody is packed into bag filter, and 4 DEG C of dialysis for 24 hours, during which change a PBS solution per 2h in 0.01M PBS；
5) antibody in bag filter is taken out, 12 4 DEG C of 000rpm centrifuge 10min, and supernatant is the antibody purified；It takes on 1 μ L
IgG content is measured with NanoDrop ultraviolet spectrometers clearly, antibody is in -80 DEG C of preservations after measurement.
6. the type and subgroup identification and titer of ascites of monoclonal antibody measure
By the anti-BALB/c mouse IgG of standard of the monoclonal antibody of purifying and Sigma companies kit1、IgG2a、IgG2b、IgG3、
IgM, κ and lambda light chain antibody carry out DAS-ELISA analyses, the results show that 11B6 monoclonal antibodies type and subclass are IgG1, kappa.
To recombinate RGDV CP albumen as envelope antigen, odd contradictive hydroperitoneum potency is detected with conventional indirect ELISA method, the results showed that 11B6
Odd contradictive hydroperitoneum potency reaches 10-8。
7. the specific detection of monoclonal antibody
With infection rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf respectively
Viral (SRBSDV), rice sawtooth dwarf virus (RRSV), rice stripe mosaic virus (RSMV) sick leaf crude extract wrap respectively
It is feminine gender with the healthy leaves crude extract of rice, the non-malicious recilia dorsalis and the non-homogenate for taking malicious rice green leafhopper taken by elisa plate
Control, sick leaf crude extract, RGDV to infect Rice Gall Dwarf In Guangdong Province, which take malicious recilia dorsalis and take the homogenate of malicious rice green leafhopper, is
Positive control measures the specificity of monoclonal antibody with ACP-ELISA methods.ACP-ELISA methods the specific steps are：Above-mentioned virus sense
Sick leaf liquid nitrogen grind into powder in mortar of dye, by 1:20 (w/v, g/mL) times are added 0.05M carbonate and are coated with buffer solution
Continue grinding homogenate, supernatant is crude extract after 5 000rpm centrifuge 3min；1 head lobe cicada is put into eppendorf centrifuge tubes, adds
With toothpick homogenate is mashed after entering 150 μ L 0.05M carbonate coating buffer solution；Crude extract or 100 holes μ L/ of homogenate are coated with ELISA
Plate；4 DEG C of overnight or 37 DEG C of 2h, make it be adsorbed in ELISA polystyrene plate holes；PBST is sealed after washing 3 times with 3% skimmed milk power
Close 30-60min；Appropriate 100 holes μ L/ of diluted monoclonal antibody, 37 DEG C of 1-2h are added；Appropriate diluted alkali is added in PBST after washing 3 times
Property phospholipase (AP, be used for rice sample) or horseradish peroxidase (HRP is used for leafhopper sample) label sheep anti-mouse igg
100 holes μ L/ of secondary antibody (Sigma companies), 37 DEG C of 1-2h；PBST is developed the color after washing 4 times with PNPP or tmb substrate, after terminating reaction
OD is read with microplate reader405Or OD450Value, with the OD value ratios of negative sample be more than 2.1 for the positive.As a result, it has been found that 11B6
Monoclonal antibody has specifically to infecting the crude extract of RGDV rice diseases leaf texture, taking malicious recilia dorsalis and carrying rice green leafhopper tissue homogenate
Property reaction, and with infection RSV, RBSDV, RRSV, SRBSDV, RSMV and healthy rice plants tissue and it is non-take malicious recilia dorsalis and
The non-homogenate for taking malicious rice green leafhopper is without any immune response.
Two, the foundation of the immunological method of detection RGDV and its kit
1. detecting the ACP-ELISA methods of RGDV
The step of 1.1 ACP-ELISA methods：
1) liquid nitrogen grind into powder in mortar is used after plant tissue is weighed, by 1:20 ratios (w/v, g/mL) are added
0.05M carbonate buffer solutions (pH 9.6) continue grinding homogenate 3min, 5 000rpm and centrifuge 3min, and 100 holes μ L/ of supernatant are added
In elisa plate, to infect RGDV rice disease leaves as positive control, healthy Rice Leaf is negative control, and 37 DEG C 2h or 4 DEG C overnight；
2) after PBST is washed 3 times 30min is closed with 3% skimmed milk power；
3) odd contradictive hydroperitoneum after appropriate dilution, 100 holes μ L/, 37 DEG C of incubation 1h are added；
4) the sheep anti-mouse igg secondary antibody of appropriate diluted alkaline phosphatase lipase (AP) label, 100 μ are added after PBST is washed 3 times
The holes L/, 37 DEG C of incubation 1h；
5) 100 holes μ L/ of PNPP nitros Phosphate substrate are added after being washed with PBST, are placed at room temperature for 30min；
6) it detects by an unaided eye, the hole that substrate colors become yellow green is the positive；Or it with after the termination reaction of 2M sodium hydroxides, uses
Enzyme-linked immunosorbent assay instrument surveys OD405, with P/N>2.1 as positive criterion.
The foundation of 1.2 ACP-ELISA methods and its sensitivity and specificity determine
The most suitable working concentration for determining monoclonal antibody and ELIAS secondary antibody in ACP-ELISA methods, i.e. ELISA are tested with conventional square formation
Plate laterally plus with the monoclonal antibody of confining liquid doubling dilution, longitudinal plus sheep anti-mouse igg secondary antibody with confining liquid doubling dilution AP labels into
Row ACP-ELISA.Experiments have shown that the most suitable working concentration of 11B6 monoclonal antibodies and AP ELIAS secondary antibodies is respectively 1:5 000 and 1:8 000
It dilutes again, the ACP-ELISA methods of RGDV in detection rice plants is established with the most suitable working concentration of antibody.It is thick to RGDV disease leaves
Extract is from 1:20 to 655 360 doubling dilutions make negative control with the healthy rice leaf crude extract of corresponding dilution respectively, point
Analyse the detection sensitivity of ACP-ELISA methods.The result shows that the ACP-ELISA methods pair 1 established:327 680 times dilution (w/v,
G/mL sick leaf crude extract) is still positive, i.e., can reach 1 to the detection sensitivity of sick leaf:327 680 (w/v, g/mL) times
Dilution shows that ACP-ELISA methods have very high sensitivity.With the ACP-ELISA methods of foundation detection RGDV, RSV,
The crude extract of RBSDV, SRBSDV, RRSV, RSMV and healthy rice plants tissue.The result shows that the ACP-ELISA methods of foundation
Detection infection RGDV rice disease leaves crude extract be in strong positive reaction, and detect respectively infect RSV, RBSDV, RRSV, SRBSDV,
RSMV and healthy rice plants tissue crude extract are negative, and yin and yang attribute Comparative result difference is extremely notable, illustrates the party
The specificity of method is very well.
The foundation and Fields detection application of 2.dot-ELISA methods
2.1 dot-ELISA methods detect the operating procedure of RGDV in plant：
Liquid nitrogen grind into powder in mortar is used after the blade of rice plants sample is weighed, by 1:20 (w/v, g/mL's)
Ratio continues grinding, homogenate after 0.01M PBS (pH7.4) are added；5 000rpm of homogenate centrifuges 3min；It takes and is checked on 2.5 μ L
Onto nitrocellulose filter (NC), while the rice leaf of health and infection RGDV are set respectively as negative and positive control；
Drying at room temperature 10-20min；NC films are immersed in the envelopes of the PBST (the 0.01M PBS containing 0.05%Tween-20) containing 5% skimmed milk power
Close 37 DEG C of closing 30min in liquid；NC films are put into the diluted monoclonal antibody of appropriateness and are incubated at room temperature 40-60min；Film is washed with PBST 3-4 times,
Each 3min；NC films are put into the sheep anti-mouse igg secondary antibody of the diluted AP enzymes label of appropriateness and are incubated at room temperature 40-60min；PBST washes film
4-5 times, each 3min；66 μ L NBT and 33 μ L BCIP substrates (Promega) are added to 10mL substrate buffer solutions (0.1mol/L
Tris Cl、0.1mol/L NaCl、0.025mol/L MgCl2, pH9.5) in mixing, film is put into chromogenic reaction 10- in substrate solution
20min, visual results wait for that positive control colour developing is apparent, and when negative no any colour developing, tap water rinse terminates anti-
It answers, photographs to record result.
The foundation of 2.2 dot-ELISA methods and its sensitivity and specificity determine
It is tested with conventional square formation and determines the monoclonal antibody of RGDV and the most suitable working concentration of ELIAS secondary antibody in dot-ELISA methods,
Experiments have shown that the most suitable working concentration of 11B6 monoclonal antibodies and ELIAS secondary antibody is respectively 1:5 000 and 1:8 000 times of dilutions.With above-mentioned anti-
The most suitable working concentration of body establishes the dot-ELISA methods of detection RGDV.Sensitivity analysis shows to be diluted to 1 when RGDV disease leaves:
When 20480 (w/v, g/mL) times, the positive spots of purple are still presented in the dot-ELISA methods detection established with 11B6 monoclonal antibodies, i.e.,
It detects the sensitivity of sick leaf and reaches 1:20480 times of dilutions (w/v, g/mL) (Fig. 1).Specificity analysis shows, this method detection
The rice plants for infecting RGDV are in strong positive reaction, and detect and infect RSV, RBSDV, RRSV, SRBSDV, RSMV and health respectively
Rice plants tissue crude extract is negative (Fig. 2).
The application of 2.3 dot-ELISA methods
It is examined with the dot-ELISA methods pair 78 plants of rice samples acquired from Rice In Guangdong field in 2017 of foundation
It surveys, as a result, it has been found that, there are 32 sample infection RGDV, incidence 41%, representative testing result such as Fig. 3 institutes in 78 samples
Show.These samples are analyzed with RT-PCR method simultaneously, the results showed that in the positive that all dot-ELISA are detected
The genetic fragment to RGDV specificity can be expanded, and is not expanded to any genetic fragment in all negative samples.PCR product core
Acid sequencing shows that positive infects RGDV really, illustrates that the dot-ELISA methods can be accurately and reliably used in plant sample
The detection of Rice Gall Dwarf In Guangdong Province.
3. Rice Gall Dwarf In Guangdong Province dot-ELISA detection kits (detection rice sample)
1) kit main component：
The above reagent is stored in 4 DEG C
2) operating procedure of rice sample is detected：
A. liquid nitrogen grind into powder in mortar is used after the blade of rice plants sample being weighed, by 1:20 ratio (w/
V, g/mL) 0.01M PBS (pH7.4) is added after continue grinding, homogenate；
B. 5 000rpm of homogenate centrifuges 3min；
C. take 2.5 μ L supernatants point samples on NC films, while the rice plants tissue that health and infection RGDV is arranged is made respectively
For negative and positive control, drying at room temperature 10-20min；
D.NC films are immersed in the PBST containing 5% skimmed milk power (the 0.01M PBS containing 0.05%Tween-20) confining liquid
Room temperature closes 30min；
E.NC films are put into 1:It is incubated at room temperature 40-60min in 4 000 times of diluted RGDV monoclonal antibodies；
F. film is washed 3-4 times with PBST, each 3min；
G.NC films are put into 1:It is incubated at room temperature 40-60min in 5 000 diluted AP enzymes label sheep anti-mouse igg secondary antibodies；
H.PBST washes film 4-5 times, each 3min；
I.66 μ LNBT and 33 μ LBCIP substrates be added to 10mL substrate buffer solutions (0.1M Tris Cl, 0.1M NaCl,
0.025M MgCl2, pH9.5) in, mixing caudacoria, which is put into substrate solution, to develop the color, visual results, waits for that positive control is obvious aobvious
Purple and negative control do not have tap water rinse film when any colour developing to terminate reaction, photograph to record result.
3) preservation and the term of validity：
It is kept in dark place in 2-8 DEG C, the term of validity 12 months.
4) buffer formulation：
A. phosphate buffer (PBS, 0.01mol/L, pH7.4)：NaCl 8g,KCl 0.2g,KH2PO4 0.2g,
Na2HPO4·12H2O 3g adjust pH to 7.4 after adding distilled water 950mL to dissolve, are settled to 1000mL.
B.ELISA cleaning solutions (0.01M PBST)：Add 0.5mL Tween-20 in 1000mL 0.01M PBS
C.ELISA confining liquids：Skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v, g/mL).
4. Rice Gall Dwarf In Guangdong Province dot-ELISA detection kits (detection recilia dorsalis or rice green leafhopper sample)
1) kit main component：
The above reagent is stored at 4 DEG C
2) operating procedure：
A. sample preparation：Single head leafhopper is put into 250 μ L eppendorf centrifugations, and often 50 μ L PBS are added in pipe, are mashed with toothpick
Leafhopper；
B. point sample：It takes a NC film to put to clean culture dish with tweezers, 3 μ L insect homogenates is taken to dub NC films
On, often take a sample to change a pipette tips；
C. it dries：Sample spot film is completed caudacoria and is dried at room temperature for, about 10-20min；
D. it closes：NC films are immersed in the closings of the PBST (the 0.01M PBS containing 0.05%Tween-20) containing 5% skimmed milk power
Room temperature closes 30min in liquid；
E. primary antibody is incubated：NC films are put into 1:It is incubated at room temperature 40-60min in 3 000 times of diluted RGDV monoclonal antibodies；
F. it washs：Film is washed with PBST 3-4 times, each 3min；
G. secondary antibody is incubated：NC films are put into 1:It is incubated at room temperature 40- in 5 000 diluted HRP enzymes label sheep anti-mouse igg secondary antibodies
60min；
H. it washs：Film is washed with PBST 4-5 times, each 3min；Finally film is washed with PBS 1 time；
I. it develops the color：It is put into new culture dish after washed NC films are blotted with filter paper, appropriate TMB assay chromogenic substrate solutions is added,
NC films are flooded, newspaper shading is covered, visual results wait for that the aobvious blue of positive control is apparent, and feminine gender when any colour developing without remembering
Record testing result, developing time about 5-15min；
3) preservation and the term of validity：
It is kept in dark place in 2-8 DEG C, the term of validity 12 months.
4) buffer formulation：
A. phosphate buffer (PBS, 0.01mol/L, pH7.4)：NaCl 8g,KCl 0.2g,KH2PO4 0.2g,
Na2HPO4·12H2O 3g adjust pH to 7.4 after adding distilled water 950mL to dissolve, are settled to 1000mL.
B.ELISA cleaning solutions (0.01M PBST)：Add 0.5mL Tween-20 in 1000mL 0.01M PBS
C.ELISA confining liquids：Skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v, g/mL).
Claims (5)
1. a kind of hybridoma cell strain 11B6 of the anti-Rice Gall Dwarf In Guangdong Province monoclonal antibody of secretion, it is characterised in that water resistant can be secreted
The specific monoclonal antibody of rice tumor dwarf virus, hybridoma cell strain 11B6 are preserved in Chinese microorganism strain guarantor on December 15th, 2017
Administration committee's common micro-organisms center is hidden, preserving number is CGMCC No.14897.
2. a kind of monoclonal antibody of the anti-Rice Gall Dwarf In Guangdong Province of hybridoma cell strain 11B6 secretions as described in claim 1.
3. the monoclonal antibody of the anti-Rice Gall Dwarf In Guangdong Province of hybridoma cell strain 11B6 secretions as claimed in claim 2, special
Sign is the odd contradictive hydroperitoneum indirect ELISA titer up to 10-8, Antibody types and subclass are IgG1, kappa, which can be with
The coat protein of Rice Gall Dwarf In Guangdong Province 47kDa has specific immune response, is analyzed using ACP-ELISA and dot-ELISA methods
It was found that the sensitivity of monoclonal antibody detection infection RGDV rice disease leaves respectively reaches 1:327 680 and 1:20 480 times of dilutions.
4. the monoclonal antibody of the anti-Rice Gall Dwarf In Guangdong Province of hybridoma cell strain 11B6 secretions as claimed in claim 2, special
Sign is that the monoclonal antibody only has a specific immune response with Rice Gall Dwarf In Guangdong Province, and with rice stripe virus, black streaked dwarf virus of rice
Poison, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, rice stripe mosaic virus and healthy rice plants and non-
It takes malicious recilia dorsalis and any immune response does not occur for non-malicious rice green leafhopper tissue of taking.
5. a kind of application of monoclonal antibody of anti-Rice Gall Dwarf In Guangdong Province as claimed in claim 2 on the viral diagnosis,
It is characterized in that the various immunological detection methods and immunological detecting kit established as core using monoclonal antibody.
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| CN109536522A (en) * | 2018-12-21 | 2019-03-29 | 贵州大学 | A kind of preparation and application of the prokaryotic expression, polyclonal antibody of southern rice black-streaked dwarf virus P6 albumen |
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